Carlos Machado - US grants

Wolbachia are maternally inherited bacteria that are found in a wide range of arthropods and filarial nematodes. The proposed research will use Neotropical fig wasps as a model system to study the dynamics and fitness effects of Wolbachia in their insect hosts. Specifically, the proposed research will investigate: 1) the population dynamics of Wolbachia, 2) the effects of Wolbachia on their hosts' reproduction, 3) the molecular evolution of Wolbachia and the effects of Wolbachia on mitochondrial and nuclear DNA variation in host populations, and 4) the ecological and evolutionary transmission of Wolbachia. Ultimately, combination of these data will provide the clearest picture yet of how transmission patterns, host population structure, and Wolbachia effects on host reproduction interact to influence Wolbachia dynamics in natural host populations.

BECAUSE OF THEIR WIDESPREAD DISTRIBUTION AND THEIR VARIED PHENOTYPIC EFFECTS AND ABILITY TO MANIPULATE THEIR HOSTS IN NUMEROUS WAYS, WOLBACHIA CURRENTLY ARE OF INTEREST TO A BROAD SPECTRUM OF BIOLOGISTS INCLUDING THOSE STUDYING HOST-PARASITE EVOLUTION, EVOLUTION OF VIRULENCE, INTRAGENOMIC CONFLICT, SELFISH GENETIC ELEMENTS, POPULATION GENETICS, AND EVOLUTIONARY BIOLOGY IN GENERAL. IMPORTANTLY, BECAUSE WOLBACHIA OCCUR IN A NUMBER OF HOST SPECIES THAT ARE ASSOCIATED WITH SERIOUS HUMAN DISEASES, INCLUDING MOSQUITOES, TSETSE FLIES, AND FILARIAL NEMATODES, THESE MICROBES MAY PROVE TO BE USEFUL AGENTS FOR BIOLOGICAL MANIPULATION OF THESE SPECIES, FOR INSTANCE BY DRIVING THE SPREAD OF GENES CONFERRING RESISTANCE TO PARASITES OR SUSCEPTIBILITY TO PESTICIDES. THE PROPOSED EXPERIMENTS WILL ADDRESS BOTH THE SHORT- AND LONG-TERM DYNAMICS OF WOLBACHIA AND MITOCHONDRIAL EVOLUTION WITHIN FIG WASPS AND WILL BE RELEVANT TO DETERMINING WHETHER WOLBACHIA CAN DRIVE SLIGHTLY DELETERIOUS, MATERNALLY INHERITED GENES INTO A POPULATION.

Understanding how new species are formed is a major question in Biology. When new species evolve from an ancestral one, their hybrids usually have several developmental or physiological problems, like infertility or lower vigor. Those problems are the result of incompatible genetic changes that have occurred in each one of the species during and after speciation. Trying to pinpoint what are those genetic changes has been difficult using traditional genetic methods. However, recently developed molecular methods that allow investigators obtain a snapshot of gene activity in all genes from an organism at any given time ("microarrays") provide an alternative, or complement, to traditional genetic methods to uncover the genetic changes underlying hybrid incompatibilities. We will use microarrays to explore this question in a classic group of fruit fly species: Drosophila pseudoobscura and its close relatives D. persimilis and D. pseudoobscura bogotana. We will investigate gene activity in hybrid sterile males from those species to first determine what genes have unusual levels of activity and will follow up only genes with unusual activity that are involved in reproduction. We will then establish whether a subset of those genes is indeed involved in hybrid sterility using traditional genetic methods (i.e. a series of genetic crosses). Finally, we will determine whether genetic changes that have occurred at those genes or at other genes that affect their activity are indeed responsible for the hybrid incompatibilities. This approach is likely to provide major insights into the identity of genes responsible for hybrid incompatibilities in these and other group of species.

The results of the proposed research will be of general interest to biologists and will allow us training students in functional genomics and evolutionary genetics. We are fully committed to integrate individuals from underrepresented groups in our research program. Finally, the identification of putative hybrid male sterility genes in Drosophila could lead to the discovery of similar genes also involved in human male infertility, and thus the health implications of the proposed research may be large.

PI: Rod A. Wing (University of Arizona)

CoPIs: Scott A. Jackson (Purdue University), Manyuan Long (University of Chicago), Carlos A. Machado (University of Maryland), and Michael J. Sanderson (University of Arizona)

Collaborators: O. Panaud (University of Perpignan, France) and D. Weigel (Max Planck Institute for Developmental Biology, Germany), Doreen Ware (Cold Spring Harbor Laboratory), Qifa Zhang and Sibin Yu (Huazhong Agricultural University, China), Bin Han (National Center for Gene Research, China), Marco Wopereis (Africa Rice Center (WARDA), Benin), Mathias Lorieux (International Center for Tropical Agriculture (CIAT), Columbia, and Georgia Eizenga (DB NRRC, Germany.

Intellectual merit. Asian cultivated rice (Oryza sativa) feeds more people than any other crop. As the rice-dependent population is expected to double in about 25 years, breeders are faced with the enormous task of doubling rice yields with less land, water, and fertilizers, and on poorer soils. It is therefore critical that the scientific community unites to provide the tools and biological/evolutionary insights required to meet future needs. The project's long-term goal is to exploit 15 million years of evolutionary diversification and adaptation that have been locked in the genomes of wild and domesticated Oryza species, to both improve cultivated rice and address fundamental questions in evolutionary and comparative genomics. To accomplish this goal the project will first generate a set of publicly available genomics resources for the genus Oryza including: i) physical maps for three wild relatives of cultivated rice and the closely related outgoup species Leersia perrieri; ii) complete sequences for the short arms of chromosome 3 of these species; iii) complete reference genome sequences for O. barthii (the wild progenitor of West African cultivated rice), and O. punctata (a wild species that will serve as the evolutionary outgroup the eight AA genome species--the species group containing O. sativa); iv) gene and small RNA expression data sets for 11 Oryza species and L. perrieri; and v) DNA polymorphism data sets across the AA genome phylogeny. The project will integrate these new resources with 5 to 8 forthcoming new Oryza reference genome sequences to create the most comprehensive within-genus comparative genomics platform for any plant system. The project will interrogate this comparative genomics platform to discover key evolutionary events and characterize the forces that led to the formation of 24 Oryza species with 10 different genome types. Specific research areas that will be addressed include: analyses of structural variation, phylogenomics, population genomics, genome evolution and the origin of new genes, and the role of transposable elements in genome evolution. Libraries, genome sequences and their annotations will be accessible via public databases and repositories, such as the AGI BAC/TEST Resource Center (http://www.genome.arizona.edu/orders/), NCBI (http://www.ncbi.nlm.nih.gov/), and Gramene (www.gramene.org).

Broader impacts. This project will have broad impacts at several levels, including basic and applied research communities, postdoctoral/graduate/undergraduate and high school student training and mentoring, community outreach, and K-5 children and their families. For basic/applied research communities, the project will enhance and develop a genus-level resource as a platform for accessing new traits and alleles that is unparalleled in the plant world. This transformative platform can be used to address fundamental hypothesis-driven questions as well as provide essential tools to improve the world's most important food crop. The project will provide training and mentoring to postdoctoral scientists, graduate/undergraduate students and high school students interested in genome evolution, plant breeding, and careers in academic and corporate science. Finally, the project will develop a biannual "Plant Science Family Night" program, targeting K-5 students and families, to get children and their families excited about plants and the role plant science plays in ensuring a safe, sustainable, and secure food supply for our planet.

The importance of non-coding RNAs is becoming increasingly apparent for a wide range of biological processes, but they are still a relatively new topic, and many aspects of their function and evolution remain poorly understood. Long intergenic non-coding (linc) RNAs are perhaps least understood among the non coding RNAs, yet they are known to play roles in dosage compensation, embryonic development, stem cell maintenance and differentiation, and epigenetic regulation of expression. The proposed work is focused on lincRNAs across the species Drosophila pseudoobscura and Drosophila persimilis. RNA-seq will be used to catalog changes in lincRNA expression during development, and these data will be used to evaluate divergence in lincRNA sequence and expression patterns.

The specific aims are the following: 1-Identify the lincRNAs in the D. pseudoobscura transcriptome using RNAseq data; 2-Characterize patterns of lincRNA expression in D. pseudoobscura and D. persimilis, with a focus on those lincRNAs that are differentially expressed; 3-Compare levels of intraspecific polymorphism to levels of interspecific divergence using expression and sequence data, and complare patterns for lincRNAs to patterns of protein-coding genes.

Broader Impacts include a summer research experience for high school students and teachers. Participant selection will be done in collaboration with minority high schools in the area, and will be facilitated by a Math and Science Partnership grant to the University System of Maryland. Undergraduate research opportunities will also be provided, and recruitment will be done partly through the University of Maryland and partly through the Smithsonian Tropical Research Institute in Panama.

Long intergenic non-coding RNAs (lincRNAs) are a recently discovered group of transcripts that have no protein-coding capacity. Although they play diverse and important roles in the biology of complex organisms, almost nothing is known of their origin and evolution. The goal of this project is to use next generation RNA sequencing technology (RNA-Seq) and the vast genomic resources of the fruit fly Drosophila to characterize the forces that underlie lincRNA sequence and expression evolution. Analyses of variation in pure species cannot explain how the regulation of lincRNA expression evolves, but analyses of misexpression in hybrids provides insight into how diverse evolutionary processes shape the regulatory process. Thus, the specific goal of this dissertation improvement grant is to analyze the evolution of lincRNA regulation using RNA-Seq in D. pseudoobscura/D. persimilis hybrids. Specific aims are (1) to identify the mode of inheritance of lincRNA expression, (2) identify whether lincRNA regulatory divergence occurs through cis or trans changes, and (3) identify whether lincRNAs may underlie hybrid incompatibilities.

This work will be the most comprehensive analysis of lincRNA evolution to date and the first to examine lincRNA regulatory evolution in closely related species via hybrids. It will underscore the importance of an understudied class of genes that deviates from the central dogma of biology. This grant will support the training of both a doctoral student and multiple undergraduates as well as provide a summer research experience for a local high school student or teacher from the University of Maryland's Minority Student Pipeline. The knowledge gleaned from this work will be disseminated widely, both in national presentations and in print. Understanding the evolution of gene regulatory systems provides important insights into the relationship between genotype and phenotype, which ultimately underlies our understanding of disease and the improvement of agricultural crops.

Biologists have recently discovered that a large fraction of the genomes of most organisms, previously thought to be "junk DNA", has biochemical activity and may be functional. Some of the genetic elements found in those regions of the genome do not code for proteins (the building blocks of cells) and have thus been named "non-coding RNAs". A few of these non-coding RNAs are known to play important roles in the biology of organisms, mainly by their capacity to turn other genes on or off. However, it is not clear if most of the non-coding RNAs that have been catalogued are even functional. This project seeks to address that gap in understanding by using patterns of evolutionary conservation in two fruit fly species to identify candidate non-coding RNAs for functional characterization. Because non-coding RNAs are found in many organisms, from flies to plants to humans, the results could have far-reaching impact. The work will be carried out by college students at graduate and undergraduate levels, thus providing them with valuable training experiences. In addition, the project will provide research experience and support with curriculum development for K-12 science teachers.

One of the most exciting recent advances in molecular biology has been the discovery of a large number of long non-coding RNAs (lncRNAs) in the transcriptomes of eukaryotic organisms. Although about a dozen lncRNAs are known to play important roles in a broad spectrum of developmental and physiological phenomena, our understanding of lncRNA function lags significantly behind our ability to quickly catalog them using RNA sequencing and computational approaches. Given that there are thousands of lncRNAs in any given genome, one essential approach to select relevant lncRNAs for functional characterization is to focus on lncRNAs with strong patterns of evolutionary conservation. This project will focus on a group of lncRNAs that are conserved between Drosophila melanogaster and D. pseudoobscura, species that diverged from their common ancestor 25-40 million years ago. These lncRNAs show small but detectable levels of DNA sequence conservation, conserved synteny, and conserved developmental expression patterns that suggest shared function in the two species. This project will characterize the phenotypic effects of that group of evolutionary conserved lncRNAs in Drosophila. Aims will include first confirming that the conserved putative lncRNAs are independent untranslated transcripts using a combination of IsoSeq and Riboseq data, then identifying their patterns of tissue-specific expression in both species using RNAseq, and finally targeting a group of the lncRNAs for functional analysis using using transcript disruption enabled by CRISPR/Cas9 genome editing in D. melanogaster. The methods developed in this study should have broad applicability across other organisms, and the functional studies should add substantially to our understanding of what lncRNAs do.

Interacting species that depend on each other for their survival and reproduction, like plants and their pollinators, have played major roles in the diversification of life on Earth. A broad goal of this project is to understand how those interacting species change and are being affected by the changing climate. The researchers will study one of the most ecologically important and highly diverse group of interacting species: figs, the small wasps that pollinate them, and the diverse group of parasitic wasps that depend on figs for survival but that in most cases do not provide benefits to them. They will study how species interactions and adaptation to local conditions change geographically, how those changes are influenced by different reproductive characteristics of the figs, and how a group of novel and diverse morphological and physiological wasp attributes that are necessary for the interaction with figs have evolved. This research will have educational impacts in K-12 student and teacher training via collaboration with public school teachers from local minority-serving high schools. Moreover, the project will have important implications for agriculture and food production because the same processes influencing this system’s responses to global change also play a role in agricultural crop-pollinator and crop-pest interactions. <br/><br/>The research has four objectives: 1) Conduct large scale geographic studies of monoecious and gynodioecious fig species to conduct comparative genomic analyses of spatial genetic structure, local adaptation, and the population genetics of species divergence on fig hosts and associated pollinating and non-pollinating fig wasps; 2) Study mechanisms of host attraction to understand their effects on fig diversification and spatial genetic structure; 3) Test the effects of environmental changes and fragmentation on the stability of mutualistic and antagonistic interactions; and 4) Study the genetic mechanisms underlying novel traits involved in synergistic and antagonistic interactions in this system. The proposed research will not only generate critical tests of theory about how host mating system constrains coevolutionary divergence and responses to environmental degradation, but will also help uncover the genetic architecture of traits involved in synergistic and antagonistic interactions between figs and their pollinating and non-pollinating wasps.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.